The healthy adult human retina contains high concentrations of blue-light absorbing carotenoid compounds in its macular region, which is the ˜1 mm diameter central retina location around the fovea. The latter is concentrated with the cone photoreceptors, which are responsible for high-acuity color vision. Known as macular pigment, MP, the carotenoids consist of three sub-species: lutein, zeaxanthin, and meso-zeaxanthin, all located in the retinal layer system anterior to the photoreceptor outer cell segments and anterior to the retinal pigment epithelium. Due to its location in the outer part of the retinal layer system, the MP is thought to shield the deeper vulnerable tissue layers from light-induced damage. This is achieved by the MP by effectively absorbing the photo-ionizing, short, deep blue to UV wavelengths of ambient light, which would otherwise reach the deeper retinal layers. The MP carotenoids are also thought to protect the tissue cells in their immediate vicinity through their well-known function as antioxidants. Much research carried out over the last two decades has investigated the role of the macular carotenoids in the prevention of Age Related Macular Degeneration (“AMD”), and more recently their role is also investigated in the improvement of visual acuity via reduction of deleterious glare effects.
Several methodologies for non-invasive optical MP assessment are currently pursued to facilitate screening of large subject populations for MP status, to track changes over time, and/or to monitor MP uptake in response to supplementation. These include the relatively widely used psychophysical method of Heterochromatic Flicker Photometry, and three emerging imaging methods permitting a quantitative objective measurement of MP levels at any macular location and in this way to obtain the MP distribution over the whole macular region. The three imaging methods include direct MP detection via Resonance Raman Spectroscopy, indirect MP detection via lipofuscin fluorescence excitation spectroscopy, also known as “autofluorescence” spectroscopy, and indirect detection via fundus reflection spectroscopy. A particular autofluoresence imaging approach is already implemented in a commercially available laser ophthalmoscopy platform that uses a 488 nm argon laser in raster-scanning mode for lipofuscin fluorescence excitation in combination with confocal detection of the fluorescence (“Model HRA”, Heidelberg Engineering, Inc., Germany); a reflection based imaging approach with conventional blue light source excitation is implemented in a commercially available fundus camera platform (“Visucam”, Zeiss Inc., Germany).
In autofluorescence spectroscopy, lipofuscin chromophores in the retinal pigment epithelial layer are excited, respectively, with wavelengths that lie within a certain blue spectral region where the absorption band of lipofuscin overlaps with the absorption band of the MP carotenoids, and with slightly longer wavelengths that still lie within the absorption region of lipofuscin but outside the absorption range of MP. This can be realized, respectively, with narrow-band 488 nm blue and 532 nm green laser light sources, with suitably filtered conventional arc-lamp or tungsten-halogen light sources, with light emitting diodes (LEDs), or with suitable sets of wavelengths provided by other light sources. Green light excitation leads to un-attenuated lipofuscin fluorescence in the macular region as well as in all peripheral regions of the retinal hemisphere. Blue light excitation, in contrast, leads to un-attenuated lipofuscin fluorescence only in the peripheral regions; in the macular region, the fluorescence intensity is now attenuated due to blue-light absorbing MP carotenoids. By comparing the lipofuscin fluorescence intensities obtained with both excitation wavelengths in foveal and peripheral retina regions, the single pass absorption of the MP can be obtained, quantified as the optical density, OD. Specifically, the MPOD is calculated for any particular location inside the macular region as the negative decimal logarithm of the ratio of the lipofuscin intensity at that macular location, Imin, to the lipofuscin intensity at peripheral locations, Imax. The lipofuscin fluorescence, which has a high oscillator strength and which occurs in a far red to near infrared broad wavelength band, is relatively easily detected in retinal (fundus) imaging configurations with a charge-coupled detector array (CCD array) under conveniently short light exposure conditions. In the healthy retina, lipofuscin is usually distributed uniformly over the retinal pigment epithelial layer. It has been shown that under this condition, two-wavelength AFI imaging with blue and green excitation light leads to the same MP results as one-wavelength AFI imaging with blue light excitation, i.e. the step of testing the uniformity of the lipofuscin distribution with the green “reference” excitation can be omitted.
Issues in the derivation of MP characteristics via autofluorescence imaging, AFI, can arise if absorption, fluorescence, and scattering effects are present due to the presence of other compounds than the lipofuscin and carotenoid chromophores of interest. Potentially these other compounds can interfere with the optical detection scheme for MP.
One issue is the fluorescence of the natural lens, which occurs at wavelengths in the visible to far red wavelength region under the autofluorescence excitation conditions described above. It has been shown that this fluorescence can be largely avoided by limiting the lipofuscin fluorescence detection to the long-wavelength shoulder of its emission band, i.e. to wavelengths above approximately 700 nm. Under these detection conditions the image contrast between lipofuscin intensities in the macular region and the periphery is significantly improved.
Another issue is the question of potential opacities in anterior ocular media, most importantly in the natural lens, where cataracts, often found in elderly subjects, could lead to AFI contrast degradation due to excessive scattering. It is necessary therefore to investigate the quantitative influence of media opacities, at least due to the predominant lens cataracts, on the achievable MP levels in AFI imaging, and possibly to derive correction factors for the AFI images in the presence of pathologies.